The present invention relates to a new compound, pharmaceutically acceptable salts thereof, compositions containing them, and their use for treating medical disorders resulting from a deficiency in growth hormone.
Growth hormone is a hormone, which stimulates growth of all tissues capable of growing. In addition, growth hormone is known to have a number of effects on metabolic processes, e.g., stimulation of protein synthesis and free fatty acid mobilisation and to cause a switch in energy metabolism from carbohydrate to fatty acid metabolism. Deficiency in growth hormone can result in a number of severe medical disorders, e.g., dwarfism.
Growth hormone is released from the pituitary. The release is under tight control of a number of hormones and neurotransmitters either directly or indirectly. Growth hormone release can be stimulated by growth hormone releasing hormone (GHRH) and inhibited by somatostatin. In both cases the hormones are released from the hypothalamus but their action is mediated primarily via specific receptors located in the pituitary. Other compounds which stimulate the release of growth hormone from the pituitary have also been described. For example arginine, L-3,4-dihydroxyphenylalanine (L-Dopa), glucagon, vasopressin, PACAP (pituitary adenylyl cyclase activating peptide), muscarinic receptor agonists and a synthetic hexapeptide, GHRP (growth hormone releasing peptide) release endogenous growth hormone either by a direct effect on the pituitary or by affecting the release of GHRH and/or somatostatin from the hypothalamus.
In disorders or conditions where increased levels of growth hormone is desired, the protein nature of growth hormone makes anything but parenteral administration non-viable. Furthermore, other directly acting natural secretagogues, e.g., GHRH and PACAP, are longer polypeptides for which reason parenteral administration is preferred.
The use of certain compounds for increasing the levels of growth hormone in mammals has previously been proposed, e.g. in EP 18 072, EP 83 864, WO 8302272, WO 8907110, WO 8901711, WO 8910933, WO 8809780, WO 9118016, WO 9201711, WO 9304081, WO 9413696, WO 9517423, WO 9514666, WO 9615148, WO 9622997, WO 9635713, WO 9700894, WO 9722620, WO 9723508, WO 9740023, and WO 9810653.
The composition of growth hormone releasing compounds is important for their growth hormone releasing potency as well as their bioavailability. It is therefore an object of the present invention to provide a novel compound with growth hormone releasing properties. Moreover, it is an object to provide a novel growth hormone releasing compound (growth hormone secretagogue) which are specific and/or selective and have no or substantially no side-effects, such as e.g. release of LH, FSH, TSH, ACTH, vasopressin, oxytocin, cortisol and/or prolactin. It is also an object to provide a compound which has a good oral bioavailability.
In accordance with the present invention there is provided a novel compound which act directly on the pituitary cells under normal experimental conditions in vitro to release growth hormone therefrom.
The growth hormone releasing compound can be utilized in vitro as unique research tools for understanding, inter alia, how growth hormone secretion is regulated at the pituitary level.
Moreover, the growth hormone releasing compound of the present invention can also be administered in vivo to increase endogenous growth hormone release.
Accordingly, the present invention relates to a new compound 2-Amino-N-[(1R)-2-[(3R)-3-benzyl-3-(N,Nxe2x80x2,Nxe2x80x2-trimethylhydrazinocarbonyl)piperidin-1-yl]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]-2-methylpropionamide, having the following chemical structure 
or a pharmaceutically acceptable salt thereof.
Furthermore, the present invention relates to the compound 2-Amino-N-[(1R)-2-[(3R)-3-benzyl-3-(N,Nxe2x80x2,Nxe2x80x2-trimethylhydrazinocarbonyl)piperidin-1-yl]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]-2-methylpropionamide 
or a pharmaceutically acceptable salt thereof.
The structure of the compound obtainable by the procedure as described in example 1 can e.g. be verified by X-ray diffraction analysis (e.g. as described in Remington: The Science and Practice of Pharmacy, 19th Edition (1995), especially pages 160 and 561-562).
Any possible combination of two or more of the embodiments described herein is comprised within the scope of the present invention.
The procedure used in this patent is based on peptide couplings well known in the art, and should in no way be interpreted as limiting the invention in any way.
In the procedure, prior to a coupling of amino acid or peptide residues, a suitable protecting group such as tert butyloxycarbonyl (Boc) can be removed with methods well known to those skilled in the art. It is also possible to avoid the use of protecting groups. The appropriate amino acids may be protected and deprotected by methods known in the art and described by e.g. T. W. Green (Protective Groups in Organic Synthesis, 2. Ed., John Wiley and Sons, New York 1991).
Example 1 describes the procedure in details. By resolution of the racemic mixture of 3-benzylpiperidine-1,3-dicarboxylic acid 1-tert-butyl ester to obtain one of the enantiomeric compounds, the final compound obtained by the procedure is the diastereomer 2-Amino-N-[(1R)-2-[(3R)-3-benzyl-3-(N,Nxe2x80x2,Nxe2x80x2-trimethylhydrazinocarbonyl)piperidin-1-yl]-1-(1H-indo-3-ylmethyl)-2-oxoethyl]-2-methylpropionamide 
instead of the mixture of the two diastereomers.
The compound of the present invention exhibits an improved resistance to proteolytic degradation by enzymes because it is non-natural, in particular because the natural amide bonds are replaced by non-natural amide bond mimetics. The increased resistance to proteolytic degradation of the compound of the invention in comparison with known hormone releasing peptides is expected to improve its bioavailability compared to that of the peptides suggested in the prior literature.
The compound of the present invention may optionally be on a pharmaceutically acceptable salt form such as the pharmaceutically acceptable acid addition salts of the compounds of the present invention which include those prepared by reacting the compound of formula I with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, mandelic phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid and/or water.
The compound of the present invention may be administered in pharmaceutically acceptable acid addition salt form or, where appropriate, as a alkali metal or alkaline earth metal or lower alkylammonium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.
In another aspect, the present invention relates to a pharmaceutical composition comprising, as an active ingredient, a compound of the present invention or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
Pharmaceutical compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington""s Pharmaceutical Sciences, 1985 or in Remington: The Science and Practice of Pharmacy, 19th Edition (1995). The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.
The pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.
Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. The amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
A typical tablet which may be prepared by conventional tabletting techniques may contain:
For nasal administration, the preparation may contain a compound of the present invention dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
Generally, the compounds of the present invention are dispensed in unit dosage form comprising 50-200 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.
The dosage of the compounds according to this invention is suitably 0.01-500 mg/day, e.g. from about 5 to about 50 mg, such as about 10 mg per dose, when administered to patients, e.g. humans, as a drug.
In a further aspect the present invention relates to a pharmaceutical composition in unit dose form, comprising as an active ingredient from about 10 to about 200 mg of the compound of the general formula I or a pharmaceutically acceptable salt thereof.
It has been demonstrated that the compound of the present invention possess the ability to release endogenous growth hormone in vivo. The compound may therefore be used in the treatment of conditions which require increased plasma growth hormone levels such as in growth hormone deficient humans or in elderly patients or livestock.
Thus, in a particular aspect, the present invention relates to a pharmaceutical composition for stimulating the release of growth hormone from the pituitary, the composition comprising, as an active ingredient, a compound of the present invention or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
In a further aspect, the present invention relates to a method of stimulating the release of growth hormone from the pituitary, the method comprising administering to a subject in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.
In a still further aspect, the present invention relates to the use of a compound of the present invention or a pharmaceutically acceptable salt thereof for the preparation of a medicament for stimulating the release of growth hormone from the pituitary.
To those skilled in the art, it is well known that the current and potential uses of growth hormone in humans are varied and multitudinous. Thus, the compound of the present invention can be administered for purposes stimulating release of growth hormone from the pituitary and would then have similar effects or uses as growth hormone itself. The compounds of the present invention is useful for: stimulation of growth hormone release in the elderly, prevention of catabolic side effects of glucocorticoids, prevention and treatment of osteoporosis, treatment of chronic fatigue syndrome (CFS), treatment of acute fatigue syndrome and muscle loss following elective surgery, stimulation of the immune system, acceleration of wound healing, accelerating bone fracture repair, accelerating complicated fractures, e.g. distraction osteogenesis, treatment of wasting secondary to fractures, treatment of growth retardation, treating growth retardation resulting from renal failure or insufficiency, treatment of cardiomyopathy, treatment of wasting in connection with chronic liver disease, treatment of thrombocytopenia, treatment of growth retardation in connection with Crohn""s disease, treatment of short bowel syndrome, treatment of wasting in connection with chronic obstructive pulmonary disease (COPD), treatment of complications associated with transplantation, treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness, treatment of obesity and growth retardation associated with obesity, treatment of anorexia, treating growth retardation associated with the Prader-Willi syndrome and Turner""s syndrome; increasing the growth rate of a patient having partial growth hormone insensitive syndrome, accelerating the recovery and reducing hospitalization of burn patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushing""s syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed patients, treatment of osteochondrodysplasias, Noonan""s syndrome, schizophrenia, depressions, Alzheimer""s disease, delayed wound healing and psychosocial deprivation, treatment of catabolism in connection with pulmonary dysfunction and ventilator dependency; treatment of cardiac failure or related vascular dysfunction, treatment of impaired cardiac function, treatment or prevention of myocardial infarction, lowering blood pressure, protection against ventricular dysfunction or prevention of reperfusion events; treatment of adults in chronic dialysis; attenuation of protein catabolic responses after major surgery, reducing cachexia and protein loss due to chronic illness such as cancer or AIDS; treatment of hyperinsulinemia including nesidioblastosis, adjuvant treatment for ovulation induction; stimulation of thymic development and prevention of the age-related decline of thymic function, treatment of immunosuppressed patients; treatment of sarcopenia, treatment of wasting in connection with AIDS; improvement in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis and renal homeostasis in the frail elderly, stimulation of osteoblasts, bone remodelling and cartilage growth; regulation of food intake; stimulation of the immune system in companion animals and treatment of disorder of aging in companion animals, promoting growth in livestock and stimulation of wool growth in sheep, increasing milk production in livestock, treatment of metabolic syndrome (syndrome X), treatment of insulin resistance, including NIDDM, in mammals, e.g. humans, treatment of insulin resistance in the heart, improvement of sleep quality and correction of the relative hyposomatotropism of senescence due to high increase in REM sleep and a decrease in REM latency, treatment of hypothermia, treatment of frailty associated with ageing, treatment of congestive heart failure, treatment of hip fractures, treatment of immune deficiency in individuals with a depressed T4/T8 cell ratio, treatment of muscular atrophy, treatment of musculoskeletal impairment in elderly, enhancing the activity of protein kinase B (PKB), improvement of the overall pulmonary function, treatment of sleep disorders, treatment of growth retardation in connection with asthma, treatment of growth retardation in connection with juvenile rheumatic arthritis, and treatment of growth retardation in connection with cystic fibrosis.
For the above indications the dosage will vary depending on the mode of administration and on the therapy desired. However, generally dosage levels between 0.0001 and 100 mg/kg body weight daily are administered to patients and animals to obtain effective release of endogenous growth hormone. Moreover the compound of the present invention has no or substantially no side-effects, when administered in the above dosage levels, such side-effects being e.g. release of LH, FSH, TSH, ACTH, vasopressin, oxytocin, cortisol and/or prolactin. Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.0001 mg to about 100 mg, preferably from about 0.001 mg to about 50 mg of the compounds of the present invention admixed with a pharmaceutically acceptable carrier or diluent.
Optionally, the pharmaceutical composition of the invention may comprise the compound of the present invention combined with one or more compounds exhibiting a different activity, e.g., an antibiotic or other pharmacologically active material.
The route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action, such as oral, nasal, pulmonary, transdermal or parenteral, the oral route being preferred.
Apart from the pharmaceutical use of the compound of the present invention, it may be useful in vitro tools for investigating the regulation of growth hormone release.
The compound of the present invention may also be a useful in vivo tool for evaluating the growth hormone releasing capability of the pituitary. For example, serum samples taken before and after administration of the compound to humans can be assayed for growth hormone. Comparison of the growth hormone in each serum sample would directly determine the ability of the patient""s pituitary to release growth hormone.
The compound of the present invention may be administered to commercially important animals to increase their rate and extent of growth, and to increase milk production.
A further use of the compound of the present invention is in combination with other secretagogues such as GHRP (2 or 6), GHRH and its analogues, growth hormone and its analogues or somatomedins including IGF-1 and IGF-2.
The compound of the present invention may be evaluated in vitro for its efficacy and potency to release growth hormone in rat pituitary primary cultures, and such evaluation may be performed as described below.
The isolation of rat pituitary cells is a modification of O. Sartor et al., Endocrinology 116, 1985, pp. 952-957. Male albino Sprague-Dawley rats (250 +/xe2x88x9225 grams) were purchased from Mollegaard, Lille Skensved, Denmark. The rats were housed in group cages (four animals/cage) and placed in rooms with 12 hour light cycle. The room temperature varied from 19-24xc2x0 C. and the humidity from 30-60%.
The rats were decapitated and the pituitaries dissected. The neurointermediate lobes were removed and the remaining tissue was immediately placed in ice-cold isolation buffer (Gey""s medium (Gibco 041-04030) supplemented with 0.25% D-glucose, 2% non-essential amino acids (Gibco 043-01140) and 1% bovine serum albumine (BSA) (Sigma A-4503)). The tissue was cut into small pieces and transferred to isolation buffer supplemented with 3.8 mg/ml of trypsin (Worthington #3707 TRL-3) and 330 mg/ml of DNase (Sigma D-4527). This mixture was incubated at 70 rotations/min for 35 min at 37xc2x0 C. in a 95/5% atmosphere of O2/CO2. The tissue was then washed three times in the above buffer. Using a standard pasteur pipette, the tissue was then aspirated into single cells. After dispersion, cells were filtered through a nylon filter (160 mm) to remove undigested tissue. The cell suspension was washed 3 times with isolation buffer supplemented with trypsin inhibitor (0.75 mg/ml, Worthington #2829) and finally resuspended in culture medium; DMEM (Gibco 041-01965) supplemented with 25 mM HEPES (Sigma H-3375), 4 mM glutamine (Gibco 043-05030H), 0.075% sodium bicarbonate (Sigma S-8875), 0.1% non-essential amino acid, 2.5% fetal calf serum (FCS, Gibco 011-06290), 3% horse serum (Gibco 034-06050), 10% fresh rat serum, 1 nM T3 (Sigma T-2752) and 40 mg/I dexamethasone (Sigma D-4902) pH 7.3, to a density of 2xc3x97105 cells/ml. The cells were seeded into microtiter plates (Nunc, Denmark), 200 ml/well, and cultured for 3 days at 37xc2x0 C. and 8% CO2.
After culturing, the cells were washed twice with stimulation buffer (Hanks Balanced Salt Solution (Gibco 041-04020) supplemented with 1% BSA (Sigma A-4503), 0.25% D-glucose (Sigma G-5250) and 25 mM HEPES (Sigma H-3375) pH 7.3) and preincubated for 1 hour at 37xc2x0 C. The buffer was exchanged with 90 ml stimulation buffer (37xc2x0 C.). Ten ml test compound solution was added and the plates were incubated for 15 min at 37xc2x0 C. and 5% CO2. The medium was decanted and analyzed for GH content in an rGH SPA test system.
The compound was tested in doses ranging from 10 pM to 100 mM. A dose-response relation was constructed using the Hill equation (Fig P, Biosoft). The efficacy (maximal GH released, Emax) was expressed in % of the Emax of GHRP-6. The potency (EC50) was determined as the concentration inducing half maximal stimulation of the GH release.
The compounds of the present invention may be evaluated for its metabolic stability using the procedure described below:
The compound is dissolved at a concentration of 1 mg/ml in water. 25 ml of this solution is added to 175 ml of the respective enzyme-solution (resulting in an enzyme:substrate ratio (w/w) of approximately 1:5). The solution is left at 37xc2x0 C. overnight. 10 ml of the various degradation solutions is analyzed against a corresponding zero-sample using flow injection electrospray mass spectrometry (ESMS) with selected ion monitoring of the molecular ion. If the signal has decreased more than 20% compared to the zero-sample, the remainder of the solution is analyzed by HPLC and mass spectrometry in order to identify the extent and site(s) of degradation precisely.
Several standard peptides (ACTH 4-10, Angiotensin 1-14 and Glucagon) have been included in the stability tests in order to verify the ability of the various solutions to degrade peptides.
Standard peptides (angiotensin 1-14, ACTH 4-10 and glucagon) were purchased from Sigma, Mo., USA)
Enzymes (trypsin, chymotrypsin, elastase aminopeptidase M and carboxypeptidase Y and B) were all purchased from Boehringer Mannheim GmbH (Mannheim, Germany)
Pancreatic enzyme mix: trypsin, chymotrypsin and elastase in 100 mM ammoniumbicarbonate pH 8.0 (all concentrations 0.025 mg/ml).
Carboxypeptidase mix: carboxypeptidase Y and B in 50 mM ammoniumacetate pH 4.5 (all concentrations 0.025 mg/ml).
Aminopeptidase M solution: aminopeptidase M (0.025 mg/ml) in 100 mM ammoniumbicarbonate pH 8.0
Mass spectrometric analysis was performed using two different mass spectrometers. A Sciex API III triple quadrupole LC-MS instrument (Sciex instruments, Thornhill, Ontario) equipped with an electrospray ion-source and a Bio-lon 20 time-of-flight Plasma Desorption instrument (Bio-lon Nordic AB, Uppsala, Sweden).
Quantification of the compound (before and after degradation) was done on the API III instrument using single ion monitoring of the molecular ion in question with flow injection of the analyte. The liquid flow (MeOH:water 1:1) of 100 ml/min was controlled by an ABI 140B HPLC unit (Perkin-Elmer Applied Biosystems Divisions, Foster City, Calif.). The instrument parameters were set to standard operation conditions, and SIM monitoring was performed using the most intense molecular ion (in most cases this corresponded to the doubly charged molecular ion).
Identification of degradation products furthermore involved the use of plasma desorption mass spectrometry (PDMS) with sample application on nitrocellulose coated targets and standard instrumental settings. The accuracy of the hereby determined masses is generally better than 0.1%.
Separation and isolation of degradation products was done using a HY-TACH C-18 reverse phase 4.6xc3x97105 mm HPLC column (Hewlett-Packard Company, Palo Alto, Calif.) with a standard acetonitril: TFA separation gradient. The HPLC system used was HP1090M (Hewlett-Packard Company, Palo Alto, Calif.).
The compound of the present invention may be evaluated for its oral bioavailability, and such evaluation may be performed as described below.
The pharmacokinetics of the compound can be investigated in fasted Beagle dogs.
Intravenous and oral administration of the test compound, in 5% glucose solution, was separated by a one weeks washout.
Blood samples were collected immediately before drug administration (time zero) and than 0.08, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, and 6.0 hours after administration.
The plasma samples were stored frozen ( less than xe2x88x9218xc2x0 C.) pending analysis.
An HPLC method with solid phase extraction and UV detection was used for the quantification of the compound in plasma.
The compound of the present invention has an oral availability of about 50%.
The pharmacokinetic parameters for compounds were calculated by non-compartmental methods using the PC based pharmacokinetic software WinNonlin, version 1.1 (Scientific Consulting Inc., Apex, N.C., USA).